This project continues to focus on two protein kinases, GCKR and GCK. These are mammalian serine/threonine protein kinases that belong to a subfamily of protein kinases, which also includes GLK, HPK1, and NIK. They are characterized by an N-terminal kinase domain related to the yeast STE20 protein kinase, a large C-terminal regulatory domain, and the ability to activate the stress-activated protein kinase (SAPK, also referred to as Jun kinase or JNK) pathway. This pathway is activated by many cellular stresses, the inflammatory cytokine tumor necrosis factor (TNF) and CD40 ligand (CD40L). CD40L is present on activated T lymphocytes and interacts with its counter-receptor CD40, which is expressed by many on the important cells in the immune system, including dendritic cells, monocytes, and B-lymphocytes. Previously we showed that GCK and GCKR are major intermediaries in TNF-mediated SAPK activation. In addition, TNF signaling leads to the recruitment of GCKR to TNF receptor associated factor-2 (TRAF-2). We have extended those studies and shown that CD40 signaling also induces GCKR activation. GCKR activation then leads to activation of the SAPK pathway. Another TNF receptor associated molecule called TANK forms a trimolecular complex with TRAF2 and GCKR. This stabilizes the interaction between TRAF2 and GCKR. Finally, we have shown that members of the Crk family of adaptor proteins bind to GCKR and thus may link GCKR to other signaling molecules. - GCK, GCKR, SAPK, TNF, CD40.